Mutant Strains of Pseudomonas Fluorescens And Variants Thereof, Methods For Their Production, And Uses Thereof In Alginate Production

ABSTRACT

It is described biologically pure bacterial cultures of mutant strains of  Pseudomonas fluorescens , which produces large amounts of alginate. The alginate may contain a certain determined content of mannuronate and guluronate residues, possible presence and determined level of acetyl groups in the alginate, and a desired molecular weight of the alginate. Also high yielding mutants with regulation of alginate production, is described. The invention further provides methods for producing new mutant strains of  Pseudomonas fluorescens  and variants thereof, and use of the resulting strains in alginate production.

FIELD OF INVENTION

This invention relates to new mutant strains of Pseudomonas fluorescens,and variants thereof, which are capable of producing large amounts ofalginate. The alginate is not only produced in large amounts, but alsowith a certain determined content of mannuronate and guluronateresidues, possible presence and determined level of acetyl groups in thealginate, and a desired molecular weight of the alginate. Also highyielding mutants with regulation of alginate production, is described.The invention further provides methods for producing new mutant strainsof Pseudomonas fluorescens and variants thereof, and use of theresulting strains in alginate production.

DESCRIPTION OF PRIOR ART

Several microorganisms are known to produce alginate, the most studiedof the bacteria is the bacterium Pseudomonas aeruginosa. It is howeverof limited use when it comes to production of alginates for use innutrients, or pharmaceuticals, because it is associated with primary andsecondary infections in mammals or humans. Other species, which might besafer sources, do not usually produce significant amounts of alginates,or alginates of sufficient high molecular weight, and can for thisreason not be used.

It is known that non-pathogenic species of Pseudomonas such as P.putida, P. mendocina and P. fluorescens produce exopolysaccharidessimilar to acetylated alginates, Govan J. R. W. et al., J. of GeneralMicrobiology (1981), 125, p. 217-220. Also Conti, E. et al.,Microbiology (1994), 140, p. 1125-1132 describe production of alginatesfrom P. fluorescens and P. putida. It is however not known any stableover-producers of alginate among these strains.

U.S. Pat. No. 4,490,467 of Kelco Biospecialties Ltd. describespolysaccharide production using novel strains of Pseudomonas mendocina.The strains produce good yields of the desired polysaccharide, and arerelatively stable in continuous fermentation. The strains are producedby exposing a wild type culture of P. mendocina with carbenicillin, andmutagenize the selected resistant mucoid clones with a mutagenic agent.The most stable and hence most preferred is deposited under the no. NCIB11687. High concentrations of alginate, approximately 20 g/l, wasobtained in nitrogen-limited continuous culture with a minimal glucosemedium. An alginate lyase activity was present in the cultures andresulted in a low molecular weight, low viscosity polymer with rheologysimilar to printing grade alginate. The degradation by the lyase enzymewas remedied with the addition of proteolytic enzyme into the medium,Hacking A. J., et al., (1983) J. Gen. Microbiol., 129, p. 3473-3480.After ten generations in continuous culture, non-mucoid variantsappeared, Sengha S. S., et al., (1989) J. Gen. Microbiol., 135, p.795-804. page 799, second paragraph.

An epimerase negative mutant of the opportunistic pathogen P. aeruginosawas reported by Chitnis et al. (1990) J. Bacteriol., 172, p. 2894-2900.Mucoid P. aeruginosa FRD1 was chemically mutagenized and mutants, whichwere incapable of incorporating guluronic acid (G)-residues intoalginate were independently isolated. Assays using G-specific alginatelyase and ¹H-nuclear magnetic resonance analyses showed that G-residueswere absent in the alginates secreted by these mutants. Goldberg andOhman, 1987, J. Bacteriol., 169, p. 1593-1602, produced up to 1.7 g/lalginate from FRD1 in shake flasks. As usual for spontaneousalginate-producers non-mucoid revertants arise frequently (Flynn andOhman, 1988, J. Bacteriol., 170, p. 1452-1460).

There is therefore still a need in the market for suitable sources forreliable alginate production in large amounts. In particular there is aneed for stable sources producing large amounts of high quality alginatewith defined structure and desired molecular weight, and especially fora source for the production of large amounts of biologically activealginate. Furthermore there is also a need for the production of puremannuronan, which can be subjected to in vitro epimerization in order toobtain alginates with a predetermined guluronate residue (G)-content.

SUMMARY OF THE INVENTION

The present invention provides new mutant strains of P. fluorescens,which are stable and produce large amounts of alginate. Some embodimentsof the invention is to provide variants thereof, which produce alginateswith a defined structure with regard to content of mannuronate andguluronate residues, possible presence of, and determined level ofO-acetyl groups and a desired molecular weight of the alginatemolecules. Also high yielding mutants with regulated alginateproduction, and methods for their production are described. Otheraspects of the invention are; methods of producing the novel mutantstrains of P. fluorescens including variants thereof, and uses of theresulting mutants in the production of alginates, in particular mediumor large-scale fermentor production of alginates, and more particularlyproduction of biologically active alginates, or pure mannuronan. Theresulting alginates are applicable in different food and industrialproducts such as nutrients, animal feedings, cosmetics andpharmaceuticals, they may also constitute an intermediate productsuitable for further modifications by mannuronan-C5-epimerases, forinstance by the epimerases of U.S. Pat. No. 5,939,289.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a biologically pure bacterial culture ofat least one mutant strain of P. fluorescens wherein said strainproduces large amounts of alginate. In a first aspect of the inventionthe said strain produces at least 10 g alginate per liter medium. Inpreferred embodiments the biologically pure bacterial culture of atleast one mutant strain of P. fluorescens produces at least 10 galginate per 40-55 g carbon source per liter medium, more preferred per50-55 g carbon source per liter medium, and most preferred thebiologically pure bacterial culture of at least one mutant strain of P.fluorescens produces at least 10 g alginate per 40 g carbon source perliter medium.

Pure mutant strain of P. fluorescens bacterium and variants thereof,covered by the invention are exemplified by mutant strains selected fromthe group consisting of the mutant strains Pf201, Pf2012, Pf2013,Pf20118, Pf20137, Pf20118algIJΔ, Pf20118algFΔ, Pf20118AlgLH203R andPf201MC. In some embodiments, the invention relates to biologically purebacterial culture of at least one strain of P. fluorescens wherein thestrain produces alginate with alginate production characteristics ofPf201 and variants thereof that retain such characteristics. Such“alginate production characteristics” may be one of more of thefollowing: yield in terms of g alginate/1 medium (g/l) and g alginate/gcarbon source (g/g carbon source), the average molecular mass, thedegree acetylation and the G-content of alginate produced.

In a second aspect the present invention comprises a pure mutant strainof P. fluorescens wherein the said mutant is capable of producing largeamounts of an alginate consisting of mannuronate residues only.Preferred variants can be selected from the group consisting of thevariant strains Pf2012, Pf2013, Pf20118, and Pf20137.

In a third aspect the present invention comprises a pure mutant strainof P. fluorescens wherein the said mutant is capable of producing largeamounts of an alginate having a defined guluronate residue (G)-contentbetween 0 and 30%. Such embodiments may be produced by methods of theinvention by exchanging, the wild type algG gene with a mutant gene, oraltering the algG gene to encode a mannuronan C-5-epimerase enzyme withlower specific activity than the wild type enzyme.

In a fourth aspect of the invention the pure mutant strain of P.fluorescens is capable of producing large amounts of an alginatewithout, or with a reduced number of O-acetyl groups. Such embodimentsmay be produced by deleting parts of, or all of the genes algI, algJ,and/or algF. The mutant variant strains Pf20118algIJΔ and Pf20118algFΔare capable of producing large amounts of an alginate without, or with areduced number of O-acetyl groups, and represents preferred embodimentsof this aspect of the invention.

In a fifth aspect of the present invention the pure mutant strain of P.fluorescens is capable of producing large amounts of an alginate with adesired molecular weight. The molecular weight of the alginate ispreferably between 50,000 and 3,000,000 Daltons. Such embodiments may beproduced by exchanging the wild type algL with a mutant gene encoding analginate lyase enzyme with lower specific activity than the wild typelyase enzyme. The pure mutant variant strain Pf20118AlgLH203R representsa preferred embodiment of the said mutant, which is capable of producinglarge amounts of an alginate with a desired high molecular weight.

In a sixth aspect of the present invention the pure mutant strain of P.fluorescens capable of producing large amounts of alginate, comprises analginate biosynthetic operon regulated by an inducible promoterdifferent from the naturally occurring promoter, and optionally one ormore effector genes. The inducible promoter is preferably a Pm promoter,and the effector gene is xylS. According to one preferred embodiment thesaid mutant strain is Pf201 MC.

A seventh aspect of the invention provides a method of producing thenovel mutant strain of P. fluorescens of the invention, wherein:

(a) a wild-type strain of P. fluorescens is contacted with a mutagenicagent, and(b) the treated bacteria of step (a) are grown in the presence of one ormore antibiotics, and(c) antibiotic resistant mucoid mutants are isolated by selection, and(d) the alginate production properties of the isolated mucoid mutants ofstep (c) are determined.The mutagenic agent of step (a) in the method is preferablynitrosoguanidine, and the antibiotics applied in step (b) is a β-lactamand/or aminoglycoside antibiotic, preferably the antibiotic iscarbenicillin. The antibiotic may be present in the range of 800-1000μg/ml medium, and more preferably in amounts of 900 μg/ml medium.

In still another aspect the present invention provides a method ofproducing a mutant strain of P. fluorescens capable of producing largeamounts of alginate where the alginate biosynthetic operon is regulatedby an inducible promoter different from the naturally occurringpromoter, and optionally one or more effector genes, wherein:

(i) the alginate biosynthetic operon promoter of a wild type strain ofP. fluorescens is exchanged by an inducible promoter by homologousrecombination, and(ii) optional effector genes are introduced into the bacterium of (i) byhomologous recombination, transposon mutagenesis or by means of aplasmid, and(iii) mutants are grown and then isolated by selection, and(iv) the alginate production properties of the isolated mutants of (iii)are determined. In one embodiment of the method according to theinvention the inducible promoter is Pm from P. putida Tol-plasmid, or amutated Pm promoter as for instance exemplified in example 9.

In still other aspects the invention comprises a method of producing amutant strain of P. fluorescens of claim 8, wherein;

-   a) the wild type algG-gene, encoding the C-5 epimerase is cloned in    a plasmid or minitransposon and mutagenized by chemical mutagenesis    or by PCR,-   b) a derivative of an alginate-producing strain of P. fluorescens,    which lacks the algG gene (ΔalgG-strain), is constructed, and-   c) the library of mutagenized algG of step (a) is transferred to the    ΔalgG-strain of P. fluorescens, and the plasmid or    transposon-containing strains were identified and assayed for    alginate-production and epimerase-activity, and-   d) the plasmid or transposon-containing strains containing a mutant    algG encoding an epimerase that provides alginate with a guluronic    acid residue content between 0 and 30% are identified by the assay    in step (c), and-   e) the mutant algG gene is cloned into a gene-replacement vector,    and-   f) the gene-replacement vector of step (e) is then transferred to an    alginate-producing strain of P. fluorescens in order to replace its    algG gene with the mutated algG gene, and making it capable of    expressing the mutant gene.

Another aspect of the invention concerns a further method of producing amutant strain of P. fluorescens of claim 8, wherein;

-   a) one or more amino acids, which is identified by mutagenesis and    subsequent screening to be important for epimerization, is    exchanged, at the gene-level, by site-specific mutagenesis to amino    acids different from the ones occurring both in the mutant and the    wild-type AlgG-protein, and-   b) the mutant gene is cloned into a gene-replacement vector and this    vector is transferred to an alginate-producing strain of P.    fluorescens where it replaces the wild-type algG gene and is capable    of being expressed.

In other aspects the invention provides use of biologically purebacterial culture of at least one mutant strain of P. fluorescens asdescribed herein for the production of alginate, and use of the alginateproduced in the preparation of a food or industrial product such as apharmaceutical, cosmetic, animal feed or nutrient product, or as anintermediate product for in vitro C-5-epimerization.

The mutant strains; Pf201, Pf2012, Pf2013, Pf20118, Pf 20137,Pf20118algFΔ, Pf20118algIJΔ, Pf20118AlgLH203R, and Pf201MC of theinvention have been deposited in The National Collections of IndustrialFood and Marine Bacteria Ltd. (NCIMB) the 16 Jul. 2002 under thefollowing accession numbers; 41137, 41138, 41139, 41140, 41141, 41142,41143, 41144 and 41145 respectively. The depositions were made inaccordance with the Budapest Treaty.

DEFINITIONS

The novel mutant strains and variants thereof of the present invention,produce alginate in large amounts, with “large amounts” as used herein,are meant at least 10 g alginate per liter. Amounts of 10 g alginate perliter medium are preferably achieved from 40-55 g carbon source perliter medium, more preferred from 50-55 g carbon source per liter mediumor most preferred from 40 g carbon source per liter medium. The alginateyields may reach 35 g alginate per liter, but amounts of about 20% to50% by weight of the carbon-source used, is more frequently achieved.

Suitable “carbon sources” can be selected from, but are not limited tomonosaccarides, disaccharides, oligosaccharides, polysaccharides,alcohols, organic acids, and are for instance fructose, glucose,galactose, sucrose, lactose, glycerol, starch, whey, molasses, sugarsirups or lactic acid (lactate), but also other C-sources as set forthin standard textbooks, such as Bergeys Manual of SystematicBacteriology, editors Noel R. Krieg and John G. Holt, 1984, Baltimore,USA might be equally used. It should be comprehended that the use ofcarbon-sources, which must not be transformed to their correspondingtriose phosphates, through the Entner-Doudoroff pathway before they canbe utilized for alginate production by the mutant bacteria, normallywill generate the highest yields, Banerjee et al., J. Bacteriol., 1983,p. 238-245. Preferably production of more than 10 g alginate/1 medium bythe mutant strains of P. fluorescens of the invention, is obtained if 40g fructose, or glycerol per liter medium is used as a carbon source. Thelarge-scale alginate production can be carried out in any suitablemanner known to a person skilled in the art, but takes preferably placein a fermentor. The fermentation is batch, fed-batch or continuous,possibly with feeding of carbon-sources and other appropriatecomponents. The fermentation is carried out at a temperature within theinterval 5-35° C. Temperatures in the lower area of this interval mightbe selected in certain cases, but preferably the fermentation is carriedout at a temperature from 20° C. to 30° C.

Selection of media, oxygenation, pH, time of fermentation, stirring, andother possible conditions of the fermentations is deemed to be withinthe general knowledge of the field, and it must be understood that avast number of combinations of two or more conditions may lead to thesame high amount of alginate yield, and that the present invention isnot limited to a specific combination of such conditions.

The mutant strains and the variants thereof, according to the presentinvention, are “stable”, that is, they do not revert to strains, whichdo not produce alginate, when they are grown over 60 generations. Themutants were grown in PIA-medium in shake flasks under standardculturing conditions as set forth in Materials and Methods, except thatthe medium was replaced with fresh PIA-medium every 24 hours (successivecultivations).

The “mutant strain” used herein comprises mutant strains of P.fluorescens Pf201, as well as variant mutant strains, which all producealginate in large amounts. In preferred embodiments, “mutant strain”refers to mutant strains of P. fluorescens Pf201 which all producealginate in large amounts. The variants might be a result of furthermutagenesis of the Pf201 mutant strain and/or further geneticengineering, or a result of genetic engineering or mutagenesis of a wildtype P. fluorescens strain. The variants will produce large amounts ofalginates of certain defined structures. Also variants containing anycombination of the herein defined mutations are considered covered bythis expression.

The alginate produced according to the invention will have a “desiredmolecular weight”. Preferably alginate with molecular weight (Mw.) inthe range from 50,000 to 3,000,000 Dalton, more preferable within200,000 to 2,000,000 Dalton, and most preferably above 300,000 Dalton,is produced.

With the expression “biologically active alginate” used herein, is meantan alginate having an impact on a biological system, i.e. certainbioactive alginate molecular structures are known to induce biologicalresponses in certain cellular systems. Such biological alginates have alower content of guluronic acid (guluronate) residues, from 0 to 30% ofthe total uronic acid content, and preferably the guluronic acid residuecontent is between 1% and 15%, and more preferably within 1% and 10%.

DESCRIPTION OF FIGURES

FIG. 1: Restriction endonuclease maps of the suicide vectors pHE55 andpMG48, confer Table 1. Only unique restriction enzyme sites shown.

FIG. 2: Growth and alginate production in fermentations with mutantstrains of P. fluorescens NCIMB 10525.

FIG. 3: ¹H-NMR-spectra of alginate produced by P. fluorescens mutantstrains Pf201 and Pf20118. The ¹H-NMR-spectra of mannuronan from theother epimerase negative mutants (Table 3) were identical with the onefor Pf20118.

FIG. 4: The alginate biosynthetic operon and the upstream open readingframe from P. fluorescens are shown. The cloned fragments are marked asboxes on the map line. Only restriction sites used for cloning areshown. The total length is 18 kb.

FIG. 5: Restriction endonuclease map of the plasmid pMC1. Only uniquerestriction enzymes are shown.

GENERAL DESCRIPTION OF MATERIALS AND METHODS Starting Materials andCulture Media Used for Growth of Bacteria

The bacterial strains, phages and plasmids used in the present inventionare listed in Table 1 below. E. coli and P. fluorescens strains wereroutinely grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, and5 g/l NaCl) or on a LA-medium, which is LB-medium containing 15 g/lagar, at 37° C. and 30° C., respectively. Pseudomonas Isolation agar(PIA, Difco) was also used for propagation of P. fluorescens. E. coliused for λ phage propagation was grown in LB-medium supplemented withmaltose (0.2%) and MgSO₄ (10 mM). Antibiotics, when used in routinegrowth experiments, were present at the following concentrations:Ampicillin 100-200 μg/ml, kanamycin 40 μg/ml, tetracycline 12.5 μg/ml(E. coli) and 30 μg/ml (P. fluorescens).

Production of P. fluorescens Alginate: Culture Media and GrowthConditions Culture media:

Production of alginate in shake flask experiments was performed inPIA-medium containing bacteriological peptone (20 g/l), MgCl₂ (1.4 g/l),NaCl (5 g/l), K₂SO₄ (10 g/l) and 87% glycerol (20 ml/l) or in PIA-mediumwith reduced salt (PIA-medium without K₂SO₄). The proteases (Alkalase2.4 l (0.15 ml/l) and Neutrase 0.5 l (0.15 ml/l)) were added to reduceextracellular alginate lyase activity, unless otherwise stated. Alkalaseand Neutrase were purchased from Novo Nordisk.

Production of alginate in fermentor was performed in PM5-mediumcontaining: fructose (40 g/l), yeast extract (12 g/l), (NH₄)₂SO₄ (0.6g/l), Na₂HPO₄x2H₂O (2 g/l), NaCl (11.7 g/l), MgSO₄x7H₂O (0.3 g/l) andclerol FBA622 (antifoam) (0.5 g/l). The proteases (Alkalase 2.4 l (0.25ml/l) and Neutrase 0.5 l (0.25 ml/l) were added to reduce extracellularalginate lyase activity.

Preparation of Standard Inoculum (Frozen Culture with Glycerol asCryoprotectant)

A colony from agar plate (incubated at 30° C. for 2-3 days, PIA-medium)is transferred to a shake flask (500 ml, baffled) with 100 ml LB-medium.The shake flask is incubated at 30° C. for 16-20 hours in an orbitalshaker (200 rpm, amplitude 2.5 cm). For preservation sterile glycerol isadded to the broth to a concentration of 15%. The mixture is transferredto sterile cryo vials (Nunc) and stored at −80° C.

Preparation of Inoculum for Production Experiments in Shake Flasks andFermentor

1 ml standard inoculum is transferred to a shake flask (500 ml, baffled)with 100 ml LB-medium. The shake flask is incubated at 30° C. for 16-20hours in an orbital shaker (200 rpm, amplitude 2.5 cm).

Alginate Production in Shake Flask

1-2 vol-% inoculum (see above) is transferred to a shake flask (500 ml,baffled) with 100 ml PIA-medium or PIA-medium with reduced salt. Theshake flask is incubated at 25° C. for 48 hours in an orbital shaker(200 rpm, amplitude 2.5 cm).

Alginate Production in Fermentor

2-3 vol-% inoculum from shake flask is transferred to a 3-literfermentor (Applicon), with 1.4 liter PM5-medium. The fermentations areperformed at 25° C. pH from start is adjusted to 7.0-7.2. pH iscontrolled at 7.0 with NaOH (2 M) and the pH-control is activated whenthe pH reaches this value. The airflow trough the culture medium is 0.25liter/liter medium (vvm) for the first 8-10 hours, thereafter it isincreased in steps up to 0.9-1.0 vvm. The dissolved oxygen is controlledat 20% of saturation by automatic control of the stirrer speed.

Applied Standard Techniques

Plasmid isolation, enzymatic manipulations of DNA and gelelectrophoresis were performed by the methods of Sambrook and Russell,2000, Molecular Cloning: A Laboratory Manual (Third Edition). ColdSpring Harbor Laboratory Press. Qiaquick Gel Extraction Kit and QiaquickPCR purification kit (Qiagen) was used for DNA-purifications fromagarose gels and enzymatic reactions, respectively. Transformation of E.coli was performed as described by Chung et al., 1989, Proc Natl AcadSci USA, 86, p. 2172-2175 or by use of heat-shock-competent rubidiumchloride cells. PCR for cloning and allele identification was performedusing the Expand High Fidelity PCR-system (Boehringer Mannheim). Astemplates were used either plasmid DNA or 1 μl of an over-night P.fluorescens culture. In the first denaturation step the reactionmixtures were heated to 96° C. for three minutes to ensure both celllysis and full denaturation of the DNA. Site-specific mutagenesis wasperformed using QuickChange Site-Directed Mutagenesis Kit (Stratagene).Primers given in Table 2 were purchased from Medprobe or fromMWG-Biotech AG. Nucleotides in the primers, which are different fromthose of the wild-type sequence are written in bold, andrestriction-enzyme sites are underlined. DNA sequencing was performedusing a Big-Dye kit (Applied Biosystems).

Construction of Suicide Vectors for Use in P. fluorescens

In order to achieve homologous recombination in P. fluorescens twodifferent suicide vectors, pHE55 and pMG48, were constructed, conferFIG. 1. The construction of pHE55 is described in Table 1. It is anRK2-based vector lacking the gene encoding TrfA, which is necessary forreplication of the plasmid. It further confers resistance to ampicillinand tetracycline, which can be used for selecting integrants. Expressionof sacB encoding levan sucrase from Bacillus subtilis has been shown tobe lethal for many gram-negative bacteria when grown on 5% sucrose (Gayet al., 1985, J. Bacteriol., 164, p. 918-921). In strain NCIMB 10525 ofP. fluorescens, however, growing non-mucoid and tetracycline resistanttransconjugants on sucrose resulted in glassy colonies, as if the strainuses the sucrose to produce a polymer. SacB and sucrose selection couldthen not be used for this strain to positively select doublecross-overs. pHE55 was used as a suicide vector in some experiments,where alginate production could be used as a marker.

The plasmid pMG48 was constructed as an alternative recombinationvector. The sacB-gene of pHE55 was replaced by a gene encoding aTrfA-LacZ-fusion protein, as described in Table 1. This protein showsβ-galactosidase activity, but the essential parts of TrfA is missing.Using plates containing XGal (5-Bromo-4-chloro-3-indolylβ-D-galactopyranoside), 60 μl of a 20 mg/ml stock solution was added toeach agar plate used for screening. The β-galactosidase activity allowfor blue/white screening both for integrants (blue colonies) and laterfor the second recombination event (white colonies).

Homologous Recombination

The DNA sequence containing the mutation of interest, either apoint-mutation, insertion or deletion together with flanking DNA of atleast 0.5 kb on each side was cloned into a suicide vector, either pHES5or pMG48. E. coli S17.1 transformed with the plasmid of interest and theP. fluorescens strain to be mutated were incubated in LB-mediumover-night. They were then incubated in fresh LB-medium, 1% inoculum wasused. E. coli was grown for two hours, P. fluorescens for four hoursprior to conjugation. One ml of each culture were then mixed andcentrifuged for 15 min. at 3000 rpm. Most of the supernatant wasremoved, and the cells were resuspended in the remaining liquid. Thedroplet containing the cells was transferred to LA-medium, and incubatedat 30° C. over-night. The cells were removed by a sterile spatula,resuspended in LB-medium, and dilutions were plated on PseudomonasIsolation agar (PIA, Difco) with appropriate antibiotics and X-Gal whenthe vector allowed for blue/white selection. A non-mucoid transconjugantcolony of each mannuronan-producing strain was incubated in 2-6sequential liquid over-night cultures in the absence of tetracycline toallow loss of the integrated plasmid. Exponentially growing cultureswere diluted 10⁴-10⁹ fold and plated on the appropriate medium to screenfor the different strains.

Measurement of G-Content and Degree of O-Acetylation of the Alginate byNMR-Spectroscopy.

Samples from fermentations were diluted in 0.2 M NaCl and centrifuged toremove the bacterial cells. For preparation of samples for determinationof degree of acetylation, alginate was precipitated from the cell freesupernatant by addition of one volume isopropanol (4° C.), andthereafter collected by centrifugation. The precipitated alginate wasthen washed twice with 70% ethanol, once in 96% ethanol, and redissolvedin distilled water before further treatment. For preparation of samplesfor determination of G-content the alginate in the cell free supernatantwas deacetylated by mild alkaline treatment as described in Ertesvåg andSkjåk-Bræk, 1999, In Methods in biotechnology 10, CarbohydrateBiotechnology Protocols. Bucke, pp 71-78. Humana Press Inc. Deacetylatedalginate was isolated from the cell free supernatant by acidprecipitation by adding HCl to pH 2. The precipitated alginate wascollected by centrifugation, redissolved in distilled water andneutralized by alkali. To reduce the viscosity of the polymer for NMRanalysis the samples were degraded by mild acid hydrolysis to a finalaverage degree of polymerisation (DPn) of about 35, that is 35 residuesin the polymer chain, neutralized and freeze dried, Ertesvåg andSkjåk-Bræk, 1999, supra. NMR-spectra were obtained using a Bruker300-MHz Spectrometer. The spectra were integrated, and the fractions ofguluronate residues (F_(G)), mannuronate block residues (F_(MM)) andalternating block residues (F_(MG=GM)) and degree of acetylation werecalculated as described in Grasdalen, 1983, Carbohydr. Res., 118, p.255-260 and Skjåk-Bræk, Grasdalen and Larsen, 1986, Carbohydr. Res.,154, p. 239-250.

Measurement of the Intrinsic Viscosity of the Alginate and DirectMeasurement of Alginate Content in Fermentation Samples.

The alginate produced was isolated, deacetylated, acid precipitated,redissolved and neutralized as described above. The neutralized alginatesolution was added isopropanol to precipitate the alginate a secondtime. The precipitated alginate was washed twice with ethanol (first 70%and then 96% ethanol), redissolved in distilled water and dialyzedagainst distilled water for 48 hours. After dialysis the sample wasfreeze dried and weighed. The intrinsic viscosity of the alginates wasdetermined on a Scott-Geräte apparatus with automatic dilution, using anUbbelodhe capillary (Φ=0.53 mm) at 20° C. and an added saltconcentration of 0.1 M NaCl. The principle of the method is as describedin Haug and Smidsrød, 1962,

Acta. Chem. Scand., 16, p1569-1578.

Enzymatic Determination of Alginate Content in Fermentation Samples.

Alginate content was measured using the M-specific lyase from abaloneand G-lyase from Klebsiella aerogenes as described by Østgaard, 1992,19, Carbohydr. Polymers, p. 51-59.

Samples from fermentations were diluted (2-20 times) in 0.2 M NaCl,centrifuged to remove bacterial cells, and deacetylated, as describedabove. The deacetylated samples were then diluted in buffer (Tris-HCl(50 mM), NaCl (0.25M), pH 7.5) to a final concentration of 0.005-0.05%alginate. LF 10/60 (FMC Biopolymer AS) or mannuronan, produced andmeasured as described herein, were used as alginate standards in theassay. For the assay one volume of sample, or standard and 0.06 volumesof alginate lyase solution (about 1 u/ml) are added to two volumes ofbuffer (Tris-HCl (50 mM), NaCl (0.25M), pH 7.5) and incubated for 3hours at 25° C. The absorbance at 230 nm is recorded before and afterthe incubation. The differences in the A230 nm values before and afterthe incubation are used for calculation of the alginate content in thesample. The results, using this assay, correlate very well with thedirect measurement of alginate content described above.

Determination of Lyase Activity

Bacterial cells from fermentations were collected by centrifugation,resuspended in buffer (Tris-HCl (50 mM), NaCl (0.25M), pH 7.5) to anoptical density of 3-10 at 660 nm and sonicated. The extracts aftersonication were investigated for lyase activity. M-specific lyase fromabalone (described by Østgaard, 1992, 19, Carbohydr. Polymers, p. 51-59)was used as standard. The lyase activities in samples were determined bymeasuring the degradation rate of mannuronan using a Scott-GeräteUbbelodhe (instrument nr. 53620/II). Mannuronan (1 mg/ml) was dissolvedin buffer (Tris-HCl (12.5 mM), NaCl (62.5 mM), pH 7.5). 4 ml ofmannuronan substrate solution and 0.4 ml of diluted standard solution,or sample were added to the Ubbelodhe capillary. The time for thesolution to pass the capillary of the Ubbelodhe was measured every 2minute over a time period of one hour. The analysis was performed at 25°C. Based on the data from the analyses, the degradation rate ofmannuronan was calculated and correlated to the lyase activity in thesample. A standard curve was obtained using the abalone M-lyase as astandard (0.005-0.05 u/ml). 1 unit of lyase activity is defined asdescribed by Ertesvåg et al., J. Bacteriol. (1998), 180, p. 3779-3784.

TABLE 1 Bacterial strains, plasmids and phages used Strains DescriptionReference E. coli recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiΔ (lac-Simon et al, S17.1 proAB), contains the necessary genes for replication1983, and transfer of RK2. Biotechnol. 1, p. 784-791. E. coli λ-pir,recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiΔ de Lorenzo et S17.1λ-pir(lac-proAB), contains the necessary genes for al, 1993, J. replicationand transfer of RK2 and replication of Bacteriol. pCB111 175, p. 6902-6907. E. coli e14- (McrA-) D(mcrCB-hsdSMR-mrr)171 endA1 Stratagene SUREsupE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC [F′proAB laclqZD(M15 Tn10 (Tetr)] E. coli XL1- Δ(mcrA) 183Δ(mcrCB-hsdSMR-mrr) 173 endA1 Stratagene Blue MRA supE44 thi-1 gyrA96relA1 lac E. coli XL1- XL1-Blue MRA (P2 lysogen) Stratagene Blue MRA(P2)P. fluorescens Non mucoid P. fluorescens wild type NCIMB NCIMB10525Pf201 algG⁺, mucoid P. fluorescens This work Pf2012 mannuronan-producingmutant, algG⁻D361N This work Pf2013 mannuronan-producing mutant, algGG430D This work Pf20118 mannuronan-producing mutant, algG R408L Thiswork Pf20137 mannuronan-producing mutant, algG S337F This workPf20118algFΔ algF in-frame deletion mutant of Pf20118. This workPf20118alglJΔ algIF in-frame deletion mutant of Pf20118. This workPf20118algLΔ algL in-frame deletion mutant of Pf20118 This workPf20118alg Pf20118-derivate encoding the AlgLH203R mutant This workLH203R protein Pf201ΔalgG algG in-frame deletion mutant This workPf20118::TnKB10 Derivative of Pf20118 with transposon from pKB10. Thiswork Pf201ΔalgG::TnKB10 Derivative of Pf201ΔalgG with transposon frompKB10. This work Pf201MC Derivative of Pf201 in which the alginatebiosynthesis This work is controlled by the inducible promoter Pm.Phages λDashII λ cloning vector Stratagene Pfλ1 λ DashII in which an 15kb insert of SauAI-partially This work digested genomic DNA from P.fluorescens NCIMB10525 containing alg′EGXLIJFA has been inserted.Plasmid pCVD442 Ori R6K, Ap^(r) Donnenberg and Kaper, 1991, 59, p.4310-4317. pJB3Tc20 RK2-based vector, Ap^(r), Tc^(r) Blatny et al.,1997, Appl. Environ. Microbiol., 63, p. 370- 379. PJB3Tc20trfADerivative of pJB3Tc20 from which a 1.0 kb BsaAI- This work NdeI-DNA-fragment encoding TrfA was deleted. pHE55 Derivative of pJB3Tc20trfAin which a 2.6 kb PstI-XbaI- This work DNA-fragment from pCVD442encoding SacB from Bacillus subtilis was inserted. pJB1002 RK2-basedvector encoding a TrfA-LacZ-fusion protein Karunakaran et al., 1998, J.Bacteriol, 180, p. 3793- 3798. pGEM5 ColE1. Ap^(R). Promega pMG47Derivative of pHE55 in which a 4.1 kb NheI-PstI-DNA- This work fragmentfrom pJB1002 encoding a TrfA-LacZ-fusion protein replaced a 2.6 kbXbaI-PstI DNA-fragment encoding SacB: pMG48 Derivative pMG47 in which a0.36 kb SphI-SapI DNA- This work fragment containing the polylinker ofpGEM5 has been inserted. pBBg10 9.9 kb Bg/II-BamHI insert from thePseudomonas Gift from A. aeuginosa alginate biosynthetic operoncontaining Chakrabarty. alg′KEGXLIJF. Ap^(r). pGEM11 ColE1. Ap^(R).Promega pMG24 pGEM11 containing a 1 kb Sa/I-DNA-fragment from This workPfλ1 encoding part of algE. pMG25 pGEM11 containing a 4.2 kb Sa/IDNA-fragment from This work Pfλ1 encoding sequences downstream of thealginate operon. pMG26 pGEM11 containing a 4.6 kb Sa/I-DNA-fragment fromThis work Pfλ1 encoding alg′GXLI′. pMG27 pGEM11 containing a 4.8 kbSa/I-DNA-fragment from This work Pfλ1 encoding alg′IJFA. pLitmus28ColE1. Ap^(R). New England Biolabs pMG23 pLitmus28 in which a 1.8 kb PCRamplified Bg/II-Pst1 This work DNA-fragment containing algG and 135 bpof algX was inserted. The primers PfalgG3r and PfalgG4f were used. pMG31Derivative of pHE55 in which an 1.8 kb Bg/II-XbaI- This work DNA-fragment encoding AlgG from pMG23 was inserted. pMG49 pMG27-derivatefrom which a 1.4 kb NruI-HpaI DNA- This work fragment was deleted,creating an in frame algI″J- deletion pMG50 pHE55 with 3441bp SacI-XbaIinsert from pMG49 This work containing algIJΔ. pMG77 Derivative of pMG27where a SacII-site was introduced This work using the primerpairalgF-SacII-1 and algF-SacII-2 (table 2). pMG78 Derivative of pMG77 fromwhich a 285 bp SacII-DNA This work fragment in algF was deleted. pMG79Derivative of SphI-SpeI-restricted pMG48 in which a This work 1.7 kbNspI-NheI-DNA fragment from pMG78 was inserted. pMG67 Derivative ofpMG26 in which an AgeI-site and an This work algLH203R was introducedusing the primers AlgLH203R1 and AlgLH203R2. pMG70 Derivative of pMG48into which a 2.5 kb PstI-NotI- This work DNA-fragement from pMG67 wasinserted into the NsiI and NotI-sites of the vector pJB658 Expressionvector containing the Pm-promoter and Blatny et al., celB xylS. Ap^(r).1997, Plasmid, 38, p. 35-51. pHE138 Derivative of pJB658celB in which a0.8 kb NdeI-NsiI- This work digested PCR-fragment encoding theN-terminal part of AlgD was inserted into the NdeI and PstI-sitesreplacing celB. pHE139 Derivative of pMG48 in which a 0.7 kbBspLUIII-SpeiI- This work digested PCR-fragment encoding the C-terminalpart of the ORF upstream of the alginate promoter was inserted into theNcoI and SpeI-sites. pHE140 Derivative of pHE138 from which a 0.6 kbNsiI-DNA- This work fragment had been removed, and the protruding endsremoved by T4-DNA-polymerase. pHE141 A Bg/II-linker was inserted intoNsiI-digested pHE139 This work which had been made blunt using T4-DNA-polymerase. pHE142 A NotI-linker was inserted downstream of xylS in Thiswork pHE140 partially digested with Eco57I. pMC1 A 2.3 kbNotl-BamHI-DNA-fragment from pHE142 was This work inserted intoNotI-Bg/II-digested pHE141. pMG51 Derivative of pMG26 where a SmaI-sitewas introduced This work at nucleotide position 368 in algG using theprimers algG-SmaI-1 and algG-SmaI-2. pMG52 Derivative of pMG51 fromwhich a 0.6 kb SmaI-DNA- This work fragment was deleted creating anin-frame deletion in algG. pMG53 Derivative of NsiI-NcoI-restrictedpMG48 in which a 2.1 This work kb Pst1-BspHI-DNA fragment from pMG52 wasinserted. pCNB111 oriR6K, mobRPA, pUT/mini-Tn5 xylS/Pm, Ap^(r), Km^(r).Winther- Larsen et al., 2000, Metabol. Eng. 2 p 79-91 pKB4 Derivative ofpMG26 from which a 3.0 kb BlpI-XhoI- This work DNA fragment was deleted.Ap^(r). 4.9 kb. pKB10 Derivative of pCNB111 in which a 1.7 kb NdeI-NotIThis work restricted PCR-fragment containing algG was inserted. pKB4 wasused as PCR-template, PfalgG-Ndel-2 and M13/pUC reverse as primers.pJT19bla Derivative of pJB655 encoding β-lactamase controlled Winther-by the Pm-promoter Larsen et al., 2000, Metabol. Eng. 2 p 92-103.pJT19D2luc Derivative of pJT19bla. Encodes the luc-gene as Winther-reporter gene Larsen et al., 2000, Metabol. Eng. 2 p 92-103. pIB11Derivative of pJT19bla containing a rrnBT1T2 Ingrid Bakke, terminatorupstream of the Pm-promoter, and the SpeI unpublished site has beenchanged to a BspLU11I-site pHH100 Derivative of pIB11 where the bla-genewas replaced This work by a luc-gene from pJT19D2luc using the enzymesNdeI and BamHI. pHH100-A2 Derivative of pHH100 containing a mutant Pm-This work promoter giving lower uninduced activity. pHH100-B1 Derivativeof pHH100 containing a mutant Pm- This work promoter giving loweruninduced activity. pHH100-D6 Derivative of pHH100 containing a mutantPm- This work promoter giving lower uninduced activity. pHH100-D9Derivative of pHH100 containing a mutant Pm- This work promoter givinglower uninduced activity. pHH100-G5 Derivative of pHH100 containing amutant Pm- This work promoter giving lower uninduced activity. pHM2Broad-host-range plasmid enoding lacOPZY from E. Mostafa et al. coli2002. Appl. Environment. Microbiol. 68: 2619-2623

TABLE 2 Primers used Name Sequence* PfalgG3r CAGGCTGCAGCACGGTTCGGCPfalgG4f AAAAAGATCTAGTCGACTCGTACATGCACC PfacetylFwCTGCTGGTGGTGATGGGCTGGG PfacetylRev AGACGCGCACGAAGCTTGAGCC algF-SacII-1GTCAAACTCGCCGCGGATCACTAC algF-SacII-2 GTAGTGATCCGCGGCGAGTTTGAC algF-1-FwAGCGATGACTTCAAGAACAACCCG algF-2-Rev CAATTTGGGTCAGAGCTACGAAGG algLH203R1AACCAACAACCGGTCCTACTGGGCCGCC3′ algLH203R2 GGCGGCCCAGTAGGACCGGTTGTTGGTTPfalgL-BspHI- AAAAAAAG TC ATGAGGTTACCTATGCAGAAGTTATTG pMG26 algG-Smal-1CACGGCATTCCCCGGGCGATCTTC algG-Smal-2 GAAGATCGCCCGGGGAATGCCGTGPfalgG-Ndel-2 AAAAAA CATATGGGAGCCTGCGCAATGAACC PfalgLRev1AAAGATCGGCAAGAACAGAAACAGG HypBspLUIII GTT ACATGT CAGCCGCAATACCTCGACCHypSpe GTT ACTAGTTTATTCGGGGGCGTGATCG AlgDNdel GGTAATTCATATGCGCATCAGCATATTTG AlgDNsil GTA ATGCATGTAGTACTGGGACAGG *The primersare written in the 5′-3′ direction. Nucleotides not found in theoriginal sequence are shown in bold. Introduced restriction-sites areunderlined.

EXAMPLES OF THE INVENTION Example 1 Preparation of Mutant Strain Pf201

The wild type P. fluorescens NCIMB10525 was purchased from The NationalCollections of Industrial Food and Marine Bacteria Ltd. (NCIMB). Thewild type does not produce significant amounts of alginate. In order toisolate alginate over-producing mutants exponentially growing cells ofP. fluorescens NCIMB 10525 were subjected to nitrosoguanidine (NG)mutagenesis. The strain was grown in nutrient broth (CM67, Oxoid) with0.5% yeast extract and washed twice in 0.1 M citrate buffer (pH 5.5)before treating the cells with 25 ug/ml nitrosoguanidine (NG) in citratebuffer for 1 hour at 30° C. The mutagenized cells were washed with 0.1 Mphosphate buffer pH 7.0 containing KH₂PO₄ (13.6 g/l) and NaOH (−2.32g/l) and inoculated (2%) into nutrient broth with yeast extract. Thecells grew overnight and were then frozen as 1 ml aliquots of NG-stock.

Dilutions of the culture were plated on PIA-medium containingcarbenicillin (900 μg/ml) and incubated at 30° C. A few mucoid mutantswere observed. From the screening, which included inspection of morethan 4*10⁵ colonies, the two most mucoid mutants were selected forfurther evaluation in fermentor studies. The better mutant, Pf201 yieldsin fermentation 11-13 g alginate per liter PM5-medium containing 40 gfructose as carbon source per liter, as depicted in FIG. 2. For growthconditions and medium composition, it is referred to Materials andMethods. The alginate produced by the Pf201 mutant using the PM5-mediumcontaining fructose, and under standard growth conditions, containsabout 30% G (guluronate residues) with complete absence of G-blocks ascan be estimated from FIG. 3. Based on the unique alginate productionproperties, the Pf201 strain was selected for further straindevelopment. The P. fluorescens mutant Pf201 of example 1 is depositedin NCIMB under the accession number 41137.

Example 2 Cloning and Sequencing of Parts of the Alginate BiosyntheticOperon

A gene library of the wild-type strain NCIMB 10525 was constructed inλDASH II (lambda Dash II) (purchased from Stratagene). Chromosomal DNAwas isolated as described by Ausubel et al., 1993, Current protocols inmolecular biology. Greene Publishing Associates, Inc and John Wiley &Sons Inc, New York. The gene-library was then constructed by insertingpartially Sau3AI-digested chromosomal DNA from NCIMB 10525 intoBamHI-digested lambda Dash II and infecting E. coli XL1-Blue MRA(P2)with the in vitro-packaged phages according to the manufacturersinstructions (Stratagene BamHI/Gigapack III Gold Extract). Labeling ofDNA-probe and detection of hybridizing λ-clones were done by use of DIGDNA Labeling and Detection Kit (Boehringer Mannheim) according to themanufacturers instructions. A 3.8 MfeI-NcoI DNA-fragment from pBBg10containing algG flanked by parts of algE and algX from P. aeruginosa waslabeled and used to screen the P. fluorescens library. One hybridizingphage, designated Pfλ1, was detected using this system and λ DNA wasisolated using Lambda Midi Kit (QIAGEN). The insert was subcloned asSalI-digested DNA-fragments into pGEM11 resulting in the four subclonespMG24-27. Sequencing of the ends of the subclones and comparison withthe alginate biosynthetic operon of P. aeruginosa revealed that Pfλ1contains the downstream part of the alginate biosynthetic operon fromthe 3′ part of algE (FIG. 4).

pMG26 and pMG27 were sequenced by Quiagen Sequencing & Genomics toobtain the full sequence of algGXLIJFA. This gene organization seems tobe similar to previously reported alginate biosynthetic clusters in; Mayand Chakrabarty, 1994, Trends Microbiol., 2, p. 151-157, Rehm et al.,1996, J. Bacteriol., 178, p. 5884-5889, Penaloza-Vazquez et al., 1997,J. Bacteriol., 179, p. 4464-4472, Vazquez et al., 1999, Gene, 232, p.217-222. The sequence has been submitted to GenBank and given theaccession number AF527790.

Example 3 Preparation of Epimerase Negative Variant Strains

The mutant strain Pf201 of Example 1 was subjected to furthermutagenesis using nitrosoguanidine using a modification of the methoddescribed in Example 1. Exponentially growing cells of P. fluorescensNCIMB 10525 were subjected to nitrosoguanidine (NG) mutagenesis: Thebacterial cells were washed twice with equal volume of Tris/maleic acid(TM) buffer pH 6.0 containing NH₄SO₄ (1.0 g/l), CaCl₂*2H₂O (4.4 mg/l),KNO₃ (6.1 mg/l), maleic acid (5.8 g/l), Tris (hydroxy methyl)-aminomethane (6.05 g/l), FeSO₄*7H₂O (0.25 mg/l) and MgSO₄*7H₂O (0.1 g/l).Cells were re suspended in 80% of original culture volume of TM-bufferand exposed to NG (50 μg/ml) for 1 hour at 30° C. The mutagenized cellswere washed with 0.1 M phosphate buffer pH 7.0 containing KH₂PO₄ (13.6g/l) and NaOH (−2.32 g/l) and inoculated (2% inoculum) into LB-mediumand incubated over-night. The death rate of the mutagenesis procedurewas calculated to approximately 90% using a non-mutagenized aliquot ofthe culture as control. After mutagenesis the culture was grown inLB-medium over-night, and dilutions of the cells plated on LA-mediumcontaining G-lyase from Kiebsiella aerogenes (about 0.1 u/dish), asdescribed by Chitnis. et al, 1990, J. Bacteriol., 172, p. 2894-2900.This G-lyase cleaves only the G-M (guluronate-mannuronate residue) andthe G-G (guluronate-guluronate residue) bonds in alginate, Haugen et al,1990, Carbohydr. Res., 198, p. 101-109.

Mucoid mutants appeared at a frequency of about 1 in 7500 on suchselective plates. One mucoid mutant was isolated and designated Pf20118.Pf20118 was grown in a fermentor under standard growth conditions usingPM5 medium, confer Materials and Methods. The polymer produced wasanalyzed by ¹H-NMR spectroscopy. The results of this analysis showedthat the mutant produced pure mannuronan, confer FIG. 3. Severalfermentations were performed with Pf20118 using standard growthconditions and the PM5-medium. Volumetric yields were in the range of14-16 g mannuronan per liter from 40 g fructose per liter medium, inapproximately 35 hours fermentations. The P. fluorescens mutant Pf20118derived from Pf201 was subject to more than 70 different experiments infermentor, none of which have indicated instability in the mannuronanproducing properties. Both Pf201 and Pf20118 have been grown for 60generations without the appearance of non-mucoid colonies. Although itseemed probable that Pf20118 had a defect in the mannuronanC-5-epimerase gene algG, it could not be excluded that the mutationsaffected other proteins, which somehow could be necessary forepimerization. A preliminary localization of the mutations responsiblefor the mannuronan producing phenotype was performed by gene-replacementof the algG allele in each of the mutants by wild-type algG. Agene-replacement vector, pMG31, encoding wild-type algG and the first135 bp of the downstream algX was constructed as described in Table 1.The plasmid was conjugated into the Pf20118 as described in Materialsand Methods using PIA containing tetracycline as selective medium.Non-mucoid colonies appeared due to the disruption of the alginatebiosynthetic operon as pMG31 recombined into algG. A non-mucoidtransconjugant colony was incubated in 2-6 sequential liquid over-nightcultures in the absence of tetracycline to allow loss of the integratedplasmid. Exponentially growing cultures were diluted 10⁻⁴-10⁹ fold andplated on PIA agar plates to screen for mucoid revertants. Mucoidcolonies were then re-streaked on L-agar containing G-lyase to test ifthey produced epimerized alginate. Such non-mucoid revertants werefound, confirming that the mutation had to be in the DNA-fragmentcorresponding to the algGX′ fragment of pMG31. The algG-gene wasamplified by PCR using the primers PfalgG3r and PfalgG4f, sequenced andthe mutation identified, confer Table 3.

Three other epimerase negative mutant derivative strains were preparedaccording to the procedure set forth above, and designated Pf2012,Pf2013, and Pf20137 respectively. They all have an identified mutationin their algG gene resulting in a different amino acid in their AlgGgene product, as set forth in Table 3 below, and this amino acid changeis sufficient to inactivate the protein. The mutants yieldedapproximately the same levels of pure mannuronan as Pf20118, when grownunder the same conditions. The epimerization defect of the mutants couldbe reverted by recombination with the wild type gene in pMG31.

TABLE 3 Mutations in algG in mannuronan-producing mutants MutantMutation in algG Amino acid substitution in gene product Pf2012G(1081)→A(1081) Asp(361) →Asn(361) Pf2013 G(1289)→A(1289) Gly(430)→Asp(430) Pf20118 C(1222)→T(1222) Arg(408) →Leu(408) Pf20137 C(−3)→T(−3)— C(1010)→T(1010) Ser(337) →Phe(337)

The mutant strains of table 3 were deposited in NCIMB under theaccession numbers; Pf2012 has the NCIMB no. 41138, Pf2013 has the NCIMBno. 41139, Pf20118 has the NCIMB no. 41140 and Pf20137 has the NCIMB no.41141.

Example 4 Preparation of Acetylase Negative and Modified VariantStrains, Pf20118algFΔ and Pf20118algIJΔ

A Pf20118 algF deletion mutant was first made, by constructing a mutantDNA-fragment containing flanking sequences of an in-frame deletion ofparts of algF, and then ligate the fragment into the suicide vectorpMG48, as described in Table 1. The resulting plasmid, designated pMG79,was transferred to P. fluorescens strain Pf20118 by conjugation asdescribed in Materials and Methods, and the transconjugants wereselected as blue colonies on PIA-plates containing XGal andtetracycline. Double recombinants were selected as white and mucoidcolonies on PIA-plates containing XGal. These candidates were furthertested for sensitivity to tetracycline. Twenty-four white, tetracyclinesensitive candidates were tested by PCR using the primer-pair algF-1-fwand algF-2-Rev as given in Table 2, and the products were analyzed bygel electrophoresis. PCR-products from twenty-two of the candidates hadthe length expected for the wild-type algF-allele (1.0 kb). However, thetwo others had the expected length for the mutant ΔalgF-allele (0.7 kb).One of these was designated Pf20118algFΔ.

A deletion mutant of algIJ was created by first creating a derivative(pMG49) of pMG27 from which a 1.4 kb NruI-HpaI DNA-fragment containingthe 261 3′ nucleotides of algI and the 5′ 1140 nucleotides of algJ wasremoved. The deletion construct encodes an in-frame fusion of AlgI andAlgJ ensuring that AlgF and AlgA should be translated normally. A 3.4 kbSacI-XbaI DNA-fragment from pMG49 was then ligated into the suicidevector pHE55 digested with the same enzymes, creating pMG50. Thisplasmid, containing the sequences flanking the deletion, was introducedto Pf20118 by conjugation from E. coli S17.1 and non-mucoidtransconjugants were selected on PlA-medium with tetracycline.Transconjugant revertants were identified as mucoid tetracyclinesensitive colonies on LA-medium. Four algIJΔ-mutant candidates weretested by PCR-amplification of a region containing the deleted regionusing the primer pair PfacetylFw and PfacetylRev (Table 2) and thePCR-product was analyzed by agarose gel electrophoresis. Two of thecolonies contained the wild type fragment (1.8 kb) while the two otherscontained the mutant segment (0.4 kb). One of these was designatedstrain Pf20118algIJΔ. Pf20118algFΔ and Pf20118algIJΔ were grown infermentors using the PM5-medium and standard growth conditions as setforth in Materials and Methods, and the produced alginate was harvestedand measured as earlier described. The results are given in Table 4below. Both variants produced mannuronan alginate in yields of 16-17 galginate per liter medium. The presence of acetyl groups was determinedby ¹H-NMR-spectroscopy as described in Materials and Methods.Pf20118algFΔ did not produce acetylated alginate, while Pf20118algIJΔproduced alginate containing small amounts of O-acetyl groups.

TABLE 4 Alginate yield, fraction of guluronate residue content [F_(G)],degree of acetylation [da] and intrinsic viscosity [η] in fermentationswith different P. fluorescens mutants Mutant Alginate (g/l) F_(G) (%) daη (dl/g) Pf201 11.3 29 0.44 16.5 Pf20118 16.0 0 0.60 17.3 Pf20118 algIJΔ16.8 0 0.03 10.9 Pf20118 algFΔ 16.2 0 0 8.9 The fermentations wereperformed in 3-I fermentors using PM5-medium and standard growthconditions. Analyzes were done as described in Material and Methods.Pf20118algFΔ and Pf20118algIJΔ are deposited in NCIMB under theaccession numbers 41142 and 41143.

Example 5 Preparation of a Modified Derivative Mutant Strains DisplayingLow Alginate Lyase Activity, Pf20118AlgLH203R

P. fluorescens has, according to current knowledge, only one alginatelyase (AlgL) encoded by the gene algL. An option for controlling themolecular weight of the alginate produced by this bacterium, istherefore to modify the AlgL gene product simultaneously produced.

The mutagenic primer pair algLH203R1/algLH203R2 was used to create aHis203Arg (H203R) mutation in the algL gene of Pf20118. The primers alsocontain silent mutations creating an AgeI-site, for alleleidentification. The mutagenic plasmid pMG70 was constructed, asdescribed in Table 1, and introduced to the Pf20118 chromosome byconjugation, and transconjugants were selected on PIA-medium withtetracycline and XGal. Transconjugants were grown in series ofover-night cultures in the absence of tetracycline and plated onPIA-medium with XGal to isolate AlgL mutants. Whitetetracycline-sensitive mutant candidates were screened byPCR-amplification of the algL-allele using the primersPfalgL-BspHI-pMG26 and PfalgLRev1 (Table 2), and alleles were identifiedby digesting the PCR-fragment with AgeI. The mutant strain chosen wasdesignated Pf20118AlgLH203R.

When the variant strain Pf20118AlgLH203R is grown in shake flasks usingthe PIA-medium with reduced salt it yields amounts of mannuronan,approximately at the same level as the variant strain Pf20118. Alsogrowth in fermentor led to approximately the same amounts of mannuronanproduced from the two variant strains (H203R produced 12 g mannuronanalginate per liter). The intrinsic viscosity measurements of themannuronan produced by Pf20118 (intrinsic viscosity of 15 dl/g) andPf20118AlgLH203R (intrinsic viscosity of 37 dl/g) in shake flasks (usingPIA medium with reduced salt, no proteases were added) show that thelatter produces a mannuronan with increased molecular weight. Pf201produced alginate with an intrinsic viscosity like Pf20118 (14 dl/g).

Bacterial cells of Pf201, Pf20118 and Pf20118AlgLH203R were harvested atthe end of the fermentation in shake flasks and sonicated. Aftersonication the extracts were investigated for alginate lyase activity,which was measured by the method as set out in Materials and methods.Defining the lyase activity of Pf201 as 100%, it is possible to detectactivities down to 2% using this method. Pf20118 showed 93% activity. Noactivity was detected for Pf20118AlgLH203R, indicating that it is lessthan 2% of that of strain Pf201. Still, when the proteases Alkalase andNeutrase, both to 0.15 ml/l were added, the intrinsic viscositiesincreased to about 50 dl/g for Pf201 and Pf20118, and to 70 dl/g forPf20118AlgLH203R, indicating that the mutant lyase has some residualactivity. The variant strain Pf20118AlgLH203R is deposited in NCIMBunder the accession number 41144.

Example 6 Preparation of Variant Mutant Strains with Reduced EpimeraseActivity

The mutant strains Pf201 and Pf20118 provide the means to make alginatein vivo, with about 30% guluronate residue content and pure mannuronan,respectively. Alginates with intermediate amounts of guluronic acid(guluronate residue) content that is between 0 and 30% can however alsobe made. One way to obtain such strains is to delete the epimerase genefrom the operon, and then introduce the gene controlled by a promotereither on a plasmid or a transposon. Plasmid pMG53 was constructed asdescribed in Table 1, this plasmid contains a variant of algG in whichthe internal 40% of the gene has been removed. This plasmid was thentransferred to Pf201 and a strain containing this deletion, designatedPf201ΔalgG, was made by homologous recombination. This strain did notmake alginate, although it did make small oligouronides containing anunsaturated residue at the non-reducing end. Plasmid pCNB111 is asuicide plasmid, which contains a mini-transposon based on Tn5. Genescan be cloned into this mini-transposon in such a way that theirexpression is controlled by the inducible Pm-promoter. The wild-typealgG-gene was transferred to this plasmid as described in Table 1,creating plasmid pKB10. The plasmid was conjugated into Pf201AalgG asdescribed in the general description of Materials and Methods underhomologous recombination. But incorporation in the chromosome was inthis case dependent on the transposon, not on homology. The resultingstrain was designated Pf201algG::TnKB10. This strain produces alginateeven in the absence of inducer, but the amount of polymer increases withincreasing concentration of inducer (not shown). When the product wasanalysed by NMR and by mass spectrometry it was found that the strainproduces a mixture of alginate and oligomers. pKB10 was also transferredto Pf 20118, creating strain Pf20118::TnKB10. This strain produces amixture of wild-type alginate containing 30% G and mannuronan (resultsnot shown). These results showed that not only is the epimerasenecessary for alginate production, it also epimerizes as part of aprotein complex. In order to obtain homogenous alginate, only one formof epimerase may be present.

A method for preparing variant strains with reduced epimerase activityis to exchange the wild type algG with a mutant gene encoding a mutantprotein with reduced activity. It is now established a method forobtaining such mutants, using the properties of Pf201ΔalgG and pKB10.Exponentially growing cells (OD_(600nm): 0.5) of E. coli S17.1 λ-pir(pKB10) were mutagenized by nitrosoguanidine. Cells from 5 ml culturewere washed twice in 5 ml TM-buffer (1.0 g/l (NH₄)₂SO₄, 0.1 g/lMgSO₄x7H₂O, 5 mg/l Ca(NO₃)₂, 0.25 mg/l FeSO₄x7H₂O, 5.8 g/1 maleic acid,6.05 g/l Tris, pH adjusted to 6.0 with NaOH). The cells were resuspended in 2.85 ml TM-buffer and treated with 100 μg/mlnitrosoguanidin for 37° C. for 30 min. The suspension were cooled on icefor three minutes, the cells were pelleted by centrifugation and washedthree times in 5 ml LB. Glycerol was added to a final concentration of10%, and the re suspension was frozen at −80° C. Only 0.007% of thecells survived the mutagenesis. The plasmids were isolated from thethawed cells and transformed into E. coli S17.1 λ-pir, of table 1. Theplasmids were now designated pKB10-M* to emphasise that they constitutea library of mutated versions of pKB10.

pKB10-M* was then conjugated into P. fluorescens Pf201ΔalgG as describedin the Materials and Methods section (under “homologous recombination”)except that 100 μl frozen E. coli λ-pir (pKB10-M*) was inoculateddirectly into 10 ml LB-medium and grown for 2 hours before being mixedwith the P. fluorescens strain. OD_(600nm) of both cultures were 0.4.After incubation on LA-medium at 30° C. for 36 hours the conjugationmixture was plated on PIA-medium (Difco) containing 40 μg/mlkanamycin(Km). Only those P. fluorescens cells in which the transposonTnKB10-M* has integrated into the chromosome will grow on this medium,because pKB10 and its derivatives are unable to replicate in P.fluorescens. Even in the absence of inducer some AlgG is expressed fromthe Pm-promoter in P. fluorescens and this level is sufficient to givemucoid colonies in strain Pf201ΔalgG::TnKB10. About 0.5% of the colonieswere non-mucoid indicating either that the algG gene in these cells wasnot expressed due to mutations in either the Pm-promoter or in themRNA-leader sequence or that the AlgG protein is non-functional.

Each plate (245×245 mm) contained 4-5000 colonies, and between 1200 and1400 of these could be picked from each plate using an automatedcolony-picker, Genetix Q-pixII, Genetix Limited, UK. The parameters wereadjusted such that the small (non-mucoid) colonies were not picked. Amethod for screening the strain containing the mutated library wasdeveloped. In this screen the parameters cell growth, alginateproduction (measured using a mixture of M- and G-lyases), and G-content(measured by using G-lyases only), respectively were measured;

Growth of Bacteria

The colonies were replicated to two different liquid media in 96 wellplates using a Genetix Q-pixII colony picker. To preserve the clones onereplica was grown in 110 μl LB-medium containing triclosan (0.025 g/l)and kanamycin(Km)(40 mg/l) and incubated for 48 hours at 25° C. Glycerol(60%, 40 μl) was added to each well, the solutions were mixed, and theplates were frozen at −80° C.

The other replicas were grown in 0.5×PIA (bacteriological pepton (10g/l), NaCl (2 g/l), MgCl₂ (0.7 g/l), K₂SO₄ (5 g/l), glycerol (5 g/l),triclosan (0.025 g/l) and Km (40 mg/l)) m-toluate (inducer) was added to0.1 mM. The bacteria were grown in sterile Nunc 96-V-well platescontaining 110 μl medium/well, and incubated for 72 hours at 25° C.using 900 rpm, orbital movement 3 mm amplitude.

Measurement of Alginate Production

NaCl (0.2 M, 120 μl) was added to each well, and the cells were pelletedby centrifugation (3900 g, 20° C., 30 min). 50 μl supernatant from eachwell were transferred to a Nunc 96-flatwell plate. The alginate wasdeacetylated by adding NaOH (0.6M, 10 μl) to each well, mixing for 25seconds, and incubating at room temperature for 1 hour. 100 μl lyasereaction buffer (50 mM Tris-HCl, 1.5% NaCl (pH 7.5)) was then added, andthe solutions were mixed for 60 seconds.

75 μl from each well was transferred to a Costar-UV 96-well plate.Another 150 μl lyase reaction buffer was added, and the solutions weremixed for 25 seconds. The absorbance at 230 nm (A1) was read, then 8 μlof G-specific alginate lyase (0.2 u/ml) was added to each well. Thesolutions were mixed for 25 seconds, and incubated at room temperaturefor 60 min. They were again mixed for 25 seconds, and read at A230 nm(A2). Then 8 μl of M-specific alginate lyase (1 u/ml) were added to eachwell, the solutions were mixed for 25 seconds and incubated at roomtemperature for 60 minutes. The solutions were again mixed for 25seconds and read at A230 nm (A3). The absorbance of the added lyases wassubtracted from the readings of A2 and A3. Alginates of knowncomposition (polymannuronic acid produced by Pf20118 and alginate with aG-content of 30% produced by Pf201) were used as standards in theassays.

The total amount of alginate is calculated based on the formula:

A _(alg) =A3−A1

By comparing A_(alg) from the samples with A_(alg) from standards withknown alginate concentration, the alginate concentration in the samplesis calculated.

Measurement of Guluronic Acid (G) Content

The relative G-content (G_(r)) in a sample is calculated by the formula

G _(r)=(A2−A1)/(A3−A1)

By comparing the G_(r) from the samples with G_(r) of the standards theG-content in the sample was calculated.

Ten thousand colonies were screened in this way, and a few candidateswith altered, but not zero, activity were picked. These mutants werethen grown in shake flasks, as described under Materials and Methods.The PIA-medium used was added triclosan (0.025 g/l), kanamycin (40 mg/l)and m-toluate (0.1 mM), and the alginate produced was analysed by NMR asdescribed in the Materials and Methods section. One mutant producedalginate containing only 13% guluronic acid residues, whereas thewild-type produces alginate containing about 30% guluronic acid residues(Table 4). Some other strains producing pure mannuronan were also found.The method makes it possible to screen mutant forms of AlgG thatintroduces less guluronic acid than the wild-type enzyme.

In order to produce further variant mutant strains producing a desiredalginate product, the mutant genes may be recovered using known PCRtechniques, cloned into pMG48, and transferred into Pf201 or actuallyany of the overproducing strains using homologous recombination, aspreviously described in the description. Similar to themannuronan-producing strains; Pf2012, Pf2013, Pf20118, and Pf20137, apoint mutation in algG affecting the epimerization is not likely toaffect the amount of alginate produced.

Example 7 Preparation of Variant Mutant Strains with Reduced EpimeraseActivity

An alternative method for preparing variant strains with reducedepimerase activity is to exchange wild type algG with a mutant geneencoding a mutant protein with less activity. Four different amino acidsubstitutions are shown in Table 3 to give epimerase negative mutants ofAlgG. In these four cases the amino acid change affect either the sizeor the charge of the amino acid, for two of them both properties arechanged. Possible additional amino acids can also be identified bysequencing mutants found by the method described in example 6.Alternative alleles of algG encoding more conservative changes in theseamino acids is made by site specific mutagenesis using pMG26 astemplate. Mutagenic primers are made which contain a codon for the newamino acid flanked by about 10-15 nucleotides identical to the knownsequence. Mutations in Ser337 will destroy the SmaI site, primers forthe other amino acids do preferably contain silent mutations introducinga restriction enzyme site to aid in identifying the new mutant strains.Primers for both strands are to be synthesized, and the mutagenesis isperformed as described in Material and Methods. Mutated algG-alleles arethen transferred to pMG48 digested with NsiI-NcoI as a 2.7 kbBspHI-PstI-digested DNA fragment. The resulting plasmids are transferredto Pf201 and transconjugants selected as being non-mucoid, tetracyclineresistant, and blue on agar plates containing XGal. After growingselected clones for several successive transfers in LB-medium, doublerecombinants are selected as having white, mucoid colonies on agarplates containing XGal, and by being sensitive to tetracycline. algGfrom these candidates can be amplified using the primer pair PfalgG5fand PfalgG3r. The amplified product is 1.7 kb long. If a restrictionsite is removed, or introduced by the primers, the correct mutants areidentified by using the corresponding restriction enzyme. Alternativelythe candidates are confirmed by DNA-sequencing.

The mutant strains are grown in shake flasks, and the alginate producedis isolated as described in Materials and Methods. The amount ofalginate and the G-content are determined using M- and G-lyases asdescribed in Materials and Methods. The results from interesting strainsare verified by NMR-spectroscopy.

Example 8 Preparation of Variant Mutant Strain Pf201MC with an InduciblePm Promoter for Regulation of the Alginate Production

The Pm promoter together with its effector protein XylS is known to be astrong inducible promoter which can be used in many gram-negativespecies, Blatny et al., 1997, 63, Appl. Environ. Microbiol. p. 370-379.The inducer used is often toluate, which diffuses freely over thebacterial membranes. The P. fluorescens strain Pf0-1 has now beensequenced at JGI (The DOE Joint GenomeInstitute)(http://spider.jgipsf.org/JGI_microbial/html/pseudomonas/pseudo_homepage.html).When the alginate operon of this strain was compared to known alginateoperon sequences from other Pseudomonas species, we found that theorganization was similar. All sequenced alginate-producing species ofPseudomonas also have the same conserved open reading frame upstream ofthe alginate promoter. It potentially encodes a protein, the function ofwhich is unknown. The objective of this experiment was to exchange thesequences downstream of the stop codon for this reading frame andupstream of the start codon of algD, the first gene in the alginateoperon, with sequences encoding XylS, the Pm-promoter and theShine-Dalgarno sequence from the vector pJB658 described in Blatny etal., 1997, 38, p. 35-51. Most of the DNA-segment, which separates xylSand the Pm-promoter in pJB658, was removed.

The first step was to clone the 3′ part of the hypothetical protein(abbreviated hyp) and the 5′ part of algD in order to get flankingsequences for the insertion. When the sequences of algEGXLIJFA of strainPf0-1 were compared to the sequences of NCIMB10525, it was found thatthe two sequences were not identical. The primers were thereforeconstructed using parts of the hyp and algD genes, which are highlyconserved in several species. The 3′ part of hyp was cloned as a 0.7 kbBspLU11I-SpeI-digested PCR fragment using the primers HypBspLU11I andHypSpeI of Table 2, into the suicide vector pMG48, generating pHE139.The 5′ part of algD was cloned as a 0.8 kb NdeI-NsiI restrictedPCR-fragment into NdeI-PstI-restricted pJB658celB, generating pHE138.The replacement vector pMC1, confer FIG. 5 was then constructed througha series of cloning steps (Table 1).

The plasmid was transferred by conjugation to strain Pf201 as describedin Materials and Methods, choosing XGal and tetracyclineresistance/sensitivity to screen for recombinants and subsequent doublerecombinants. Colonies, which seemed to be more mucoid on PIA-mediumcontaining 1 mM toluate, than on PIA-medium not containing toluate, werechosen for further analyses by PCR. Using the primer pair HypBspLU11Iand AlgDNsiI (Table 2) the expected PCR-product from wild type strainswould be 2.3 kb long, while that of the mutant strain would be 3.0 kb.The chosen mutant was designated Pf201MC. This strain was then fermentedin the absence and presence of toluate (0.025 mM) as an inducer.PM5-medium and standard conditions were used during the fermentation, asdescribed in Material and Methods. The un-induced culture produced 3.5 galginate per liter medium, whereas the induced culture produced 13 galginate per liter medium. The mutant strain Pf 201MC is deposited inNCIMB under the accession number 41145.

Example 9 Use of an Inducible Mutated Pm Promotor for Regulation ofAlainate Production

The wild type Pm-promoter does function, however the un-induced level ofexpression is fairly high. It has been developed a method to screen formutations in the said promoter, based on the work of Winther-Larsen etal., Metabol. Eng. (2000), 2, p 92-103. The original pJT19-bla waschanged by inserting a terminator sequence and an AflIII-site upstreamof the Pm-promoter and the SpeI-site downstream of the promoter waschanged into a BspLU11I-site. The new plasmid was designated pIB11. Inthis plasmid the Pm-promoter is flanked by unique XbaI and BspLU11Irestriction sites. Two complementary 50 bp DNA-oligomers covering thisDNA-fragment were then synthesised. The conditions were chosen to givean error rate of about 12% over the nucleotides flanked by theserestriction sites. The corresponding wild-type strands were alsosynthesised. A library of double-stranded oligonucleotides was then madeby annealing each of the oligonucleotides containing mutations with thecomplementary wild-type oligonucleotide. The ends of theoligonucleotides were constructed to be complementary to pIB11restricted with XbaI and BspLU11I. The annealed oligonucleotides werethen ligated into pIB11 restricted with XbaI and BspLU11I, and 50 000transformants were obtained. In E. coli this library might be screenedusing resistance for ampicillin as a marker. But P. fluorescens alreadyhas a fairly high resistance towards β-lactams. The gene for β-lactamasewas exchanged with a gene encoding luciferase as described in Table 1,creating the vectors pHH100 containing the wild-type-promoter andpHH100-library containing the library of promoters. The pHH100-librarycontains 8000 independent transformants. It was then transferred to P.fluorescens by conjugation as described in Materials and Methods.

The library of mutated Pm-promoters was screened for luciferase activityusing the assay described by Wood, K. V. and DeLuca, M. (1987, Anal.Biochem. 161: 501-507). To obtain reproducible results we found that thebacteria first had to be grown in microtiter plates containing 110 μlliquid PIA-medium with 40 μg/ml kanamycin (Km) (25° C., 48 hours,shaking at 900 rpm). Some bacteria were then diluted in new medium,using a sterile stamp for transfer, and then stamped onto two nylonfilters. The filters were placed on PIA-plates with and without inducer(1 mM m-toluate), the bacteria facing up, and incubated for 14 hours at30° C. The filter was then placed in a Petri dish containing 3 mlluciferin (Promega), (1 mM in 0.1 M sodium citrate, pH 5.0), shakenuntil the liquid was distributed evenly, and incubated for 10 minutes.It was placed on a filter-paper to remove the liquid, and placed, facedown, on transparent plastic film. A dry filter paper was placed on topof it to remove residual dampness. The nylon filter was then exposed for10 min. using a Kodak 2000IR camera. 1200 colonies were screened by thismethod, and 84 were identified that showed no or only weak activity fromcolonies grown without inducer and readily detectable activity fromcolonies grown in the presence of inducer. Seventy-nine of thesecolonies were re-screened, and seventy-five of them showed significantlylower activity in the absence of inducer compared to the wild-typepHH100.

Six of these clones were grown in 10 ml LB containing 50 mg/l kanamycin.Five were chosen because their expression levels without inducer wasvery low, the sixth (Pf201 (pHH100-E1)) had an intermediary level ofexpression without added inducer, but also a significantly higherexpression level in the presence of inducer. Stationary phase cultures(100 μl) were then transferred to two shake flasks containing 10 mlfresh LB-medium and incubated for two hours before adding m-toluate to afinal concentration of 1 mM. The cultures were harvested 14 hours afterinduction. Ninety μl culture were then added to 1.5 ml tubes containing10 μl buffer (1 M K₂HPO₄, 20 mM EDTA, pH 7.8) and frozen at −80° C.Luciferase activity was measured using the Luciferase assay system fromPromega Inc (Cat. nr. E1500) (Table 5). The method proved to be usefulto find mutant promoters achieving not only a very low un-inducedexpression level, but also having a low un-induced expression level andstill a fairly high induced expression level compared to the wild type.The results are given in the table below.

TABLE 5 Luciferase activities from Pm expression in P.fluorescens-mutants Uninduce cells Induced cells Clone Activity^(a)%^(b) Activity^(a) %^(b) Ratio^(c) NCIMB10525 11.6 100 605 100 52(pHH100) NCIMB10525 0.7 6.0 3.2 0.5 5 (pHH100-A2) NCIMB10525 1.3 11 5.70.9 4 (pHH100-B1) NCIMB10525 1.2 11 15 2.4 12 (pHH100-D6) NCIMB10525 0.54.2 8.7 1.4 18 (pHH100-D9) NCIMB10525 7.8 67 1050 173 134 (pHH100-E1)NCIMB10525 0.5 4.3 69 11.4 138 (pHH100-G5)Strain NCIMB10525 was used as blank, and had no measurable activity. Theaverage results from two independent inoculations of each strain areshown. a: The activity is given in arbitrary units (the values aredependent on the settings of the instrument). b: Activity in percent ofpercent of wild-type levels. C: Induced/un-induced values.

Example 10 In Vitro Epimerization of Mannuronan Alginate Product

Mannuronan produced as described in example 4, was dissolved in buffer(Mops (50 mM), CaCl₂ (2.5 mM), NaCl (10 mM), pH 6.9) to a concentrationof 0.25% mannuronan alginate. The mannuronan C5-epimerase AlgE4 producedand purified as described by Ramstad et al, Enzyme and MicrobialTechnology, (1999), 24, p 636-646, was added to a concentration of 1 mgenzyme/200 mg mannuronan. The solution was incubated at 37° C. for 23hours. The epimerization was stopped by acid precipitation of thealginate. The alginate was then re-dissolved in distilled water andneutralized with alkali. The alginate solution was added NaCl to aconcentration of 0.2% and one volume of ethanol (96%) to precipitate thealginate. The precipitated alginate was washed 3 times with 70% ethanol,and 2 times with 96% ethanol, and freeze dried. The freeze-driedalginate was treated further and analyzed by NMR as described inMaterials and Methods. The product after this incubation was an almosttotally poly alternating alginate (PolyMG, Table 6).

Poly MG was re-dissolved in buffer (Mops (50 mM), CaCl₂ (2.5 mM), NaCl(10 mM), pH 6.9). The mannuronan C5-epimerase AlgE1 was produced andpurified as described by Ramstad et al, Enzyme and Microbial Technology,(1999), 24, p 636-646, except that it was purified by ion-exchangechromatography only. It was added to a concentration of 1 mg enzyme/200mg mannuronan. The solution was incubated at 37° C. for 4 days.Additional AlgE1 (1 mg enzyme/200 mg mannuronan) was added after 1, 2and 3 days incubation. The epimerization was stopped as described above.Alginate was isolated and analyzed by NMR as described above. The resultof the epimerization was an alginate with a G-content of >95% (Table 6).

TABLE 6 Composition of alginate after epimerization of mannuronan withAlgE4 and AlgE1 Alginate FG FM FGG FGM/MG FMM Mannuronan 0 1.0 0 0 1.0PolyMG 0.45 0.55 0 0.45 0.1 (mannuronan + AlgE4) Poly G >0.95 <0.05 >0.9<0.05 <0.02 (PolyMG + AlgE1)

Example 11 Alginate Production Utilizing Different Carbon Sources

Pseudomonades are known to have the ability to utilize numerouscompounds for growth. In this example the ability of metabolisingdifferent carbon sources to alginate is demonstrated using thePM5-medium replacing fructose with the actual carbon source. The mediumcomposition, growth conditions and analyses of alginate concentrationsin these fermentation experiments were performed as described in“General description of Materials and Methods”, unless otherwise stated.

The ability to produce alginate is demonstrated for an alcohol(glycerol), monosaccharides (fructose, glucose) and for a disaccharide(lactose) (see Table 7).

P. fluorescens does not encode β-galactosidase, and is therefore unableto use lactose as a carbon source even though it can grow on bothglucose and galactose. We transferred the plasmid pHM2 encoding E. coliβ-galactosidase (LacZ) and lactose permase (LacY), as described byMostafa, H. E., Heller, K. J., Geis, A. 2002. Appl. Environment.Microbiol. 68: 2619-2623, to strain Pf201 by conjugation as described inthe chapter of homologous recombination in the description. Theresulting strain is denominated Pf201 (pHM2). To avoid problems ofplasmid loss, lacZ and lacY could preferably be inserted into thechromosome using derivatives of plasmid pCNB111. Whey, a waste productfrom the production of cheese, contains high amounts of lactose. WhenPf201 (pHM2) was grown in PM5-medium containing 27.5% ultrafiltratedwhey, corresponding to 50.9 g/l lactose in the medium, 13.3 g/l alginatewas produced.

The results obtained are given in table 7 and indicate that numerouscarbon sources can be utilized to yield large amounts of alginate by thealginate overproducing mutant strains.

TABLE 7 Production of alginate by utilization of different carbonsources (C-source) Volumetric C-yield Amount of yield of (g alginate/gStrain C-source C-source (g/l) alginate (g/l) C-source) Pf20118Fructose¹⁾ 40 16.0 0.40 Pf20118 Glucose²⁾ 40 13.0 0.33 Pf20118Glycerol¹⁾ 40 17.1 0.43 Pf201(pHM2) Lactose³⁾ 51 13.3 0.26 ¹⁾All of thecarbon source (40 g/l) is added before inoculation of the fermentation.²⁾4.5 g/l glucose is added to the medium before the start of thefermentation. The rest of the glucose (35.5 g/l) is fed at a continuousrate (1 g glucose/liter, hour). The glucose-feeding is started 10 hoursafter inoculation ³⁾Ultrafiltrated whey containing as main compoundlactose and minerals as minor compounds are added to the PM-5 medium.The ultrafiltrated whey also contains traces of milk proteins. Thelactose concentration in the medium was 51 g/l after the addition of theultrafiltrated whey. The ultrafiltrated whey was added beforeinoculation of the fermentations.

1-26. (canceled)
 27. A mutant strain of P. fluorescens which comprises amutant algG gene and produces alginate having a defined guluronateresidue (G)-content between 0 and 30%, wherein said algG gene isinactivated or encodes an enzyme having reduced epimerase activity. 28.A mutant strain of P. fluorescens which produces alginate, wherein insaid mutant strain the alginate biosynthetic operon has been placedunder the control of an inducible promoter which replaces the nativepromoter of said operon.
 29. The mutant strain of P. fluorescens ofclaim 28, wherein said mutant strain produces at least 10 g alginate perliter medium.
 30. The mutant strain of P. fluorescens of claim 28,wherein the inducible promoter is a Pm promoter or a mutant thereof. 31.The mutant strain of P. fluorescens of claim 28, wherein the induciblepromoter is a Pm promoter, and the mutant strain further comprises anxylS gene.
 32. The mutant strain of P. fluorescens of claim 28, whereinthe inducible promoter is a Pm promoter from the Pseudomonas putida TOLplasmid.
 33. The mutant strain of P. fluorescens of claim 28, whereinthe said mutant produces an alginate consisting of mannuronate residuesonly.
 34. The mutant strain of P. fluorescens of claim 28, wherein thesaid mutant produces alginate having a defined guluronate residue(G)-content between 0 and 30%.
 35. The mutant strain of P. fluorescensof claim 28, wherein the said mutant produces alginate without, or witha reduced number of O-acetyl groups.
 36. The mutant strain of P.fluorescens of claim 28, wherein the said mutant produces alginate witha molecular weight of between 50,000 and 3,000,000 Daltons.
 37. Themutant strain of P. fluorescens of claim 28, wherein the mutant strainfurther comprises a mutant gene selected from the group consisting: amutant algG gene, a mutant algI gene, a mutant algJ gene, a mutant algLgene, and a mutant algF gene.
 38. The mutant strain of P. fluorescens ofclaim 28, wherein the mutant strain further comprises a mutant algG genewhich encodes an epimerase enzyme having reduced epimerase activity. 39.The mutant strain of P. fluorescens of claim 28, wherein the mutantstrain further comprises a mutant algG gene which is inactivated.
 40. Abiologically pure bacterial culture of the mutant strain of P.fluorescens of claim
 28. 41. A mutant strain of P. fluorescens, whereinsaid strain produces at least 10 g alginate per liter medium.
 42. Themutant strain of claim 41 which has a mutation corresponding to themutation in Pseudomonas fluorescens mutant strain Pf201 and which isstable over at least 60 generations.
 43. The mutant strain of claim 41,wherein the mutant strain further comprises a mutant gene selected fromthe group consisting of: a mutant algG gene, a mutant algI gene, amutant algJ gene, a mutant algL gene and a mutant algF gene.
 44. Amethod of producing a mutant strain of P. fluorescens of claim 41,wherein: a) a wild-type strain of P. fluorescens is contacted with amutagenic agent, and b) the treated P. fluorescens of step (a) are grownin the presence of one or more antibiotics, and c) antibiotic resistantmucoid mutants are isolated by selection, and d) the alginate productionproperties of the isolated mucoid mutants of step (c) are determined.45. The method according to claim 44, wherein the mutagenic agent isnitrosoguanidine.
 46. The method according to claim 44, wherein thetreated P. fluorescens of step (a) are grown in the presence of aβ-lactam or aminoglycoside antibiotic.
 47. The method according to claim44, wherein the treated P. fluorescens of step (a) are grown in thepresence of carbenicillin.
 48. A method of producing a mutant strain ofP. fluorescens of claim 28, wherein (i) the alginate biosynthetic operonpromoter of a wild type strain of P. fluorescens is exchanged by aninducible promoter by homologous recombination, and (ii) optionaleffector genes are introduced into the bacterium of (i) by homologousrecombination, transposon mutagenesis or by means of a plasmid, and(iii) mutants are grown and then isolated by selection, and (iv) thealginate production properties of the isolated mutants of (iii) aredetermined.
 49. The method according to claim 48, herein the induciblepromoter is Pm from P. putida Tol-plasmid, or a mutated Pm promoter. 50.A method of producing a mutant strain of P. fluorescens of claim 27,wherein a) the wild type algG-gene, encoding the C-5 epimerase is clonedin a plasmid or minitransposon and mutagenized by chemical mutagenesisor PCR, b) a derivative of an alginate-producing strain of P.fluorescens, which lacks the algG gene (ΔalgG-strain), is constructed,and c) the library of mutagenized algG of step (a) is transferred to theΔalgG-strain of P. fluorescens, and the plasmid or transposon-containingstrains are identified and assayed for alginate-production andepimerase-activity, and d) the plasmid or transposon-containing strainsof a mutant algG encoding an epimerase that provides alginate with aguluronic acid residue content between 0 and 30% are identified by theassay in step (c), and e) the mutant algG gene is cloned into agene-replacement vector, and f) the gene-replacement vector of step (e)is then transferred to an alginate-producing strain of P. fluorescens inorder to replace its algG gene with the mutant algG gene, and making itcapable of expressing the mutant gene.
 51. A method of producing amutant strain of P. fluorescens of claim 27, a) one or more amino acids,which are identified by mutagenesis and subsequent screening to beimportant for epimerization, are exchanged, at the gene-level, bysite-specific mutagenesis to amino acids different from the onesoccurring both in the mutant and the wild-type AlgG-protein, and b) themutant gene is cloned into a gene-replacement vector and this vector istransferred to an alginate-producing strain of P. fluorescens where itreplaces the wild-type algG gene and is capable of being expressed. 52.A method of producing alginate comprising culturing at least one mutantstrain of P. fluorescens of claim 27 under conditions sufficient toproduce alginate.
 53. A method of large scale fermentor production ofalginate comprising culturing at least one mutant strain of P.fluorescens of claim 27 under conditions sufficient for large scalefermentor production of alginate.
 54. A method of producing alginatecomprising culturing at least one mutant strain of P. fluorescens ofclaim 28 under conditions sufficient to produce alginate.
 55. A methodof large scale fermentor production of alginate comprising culturing atleast one mutant strain of P. fluorescens of claim 28 under conditionssufficient for large scale fermentor production of alginate.
 56. Acomposition comprising the alginate produced by the mutant strain of P.fluorescens of claim 33.